1. Field of the Invention
The present invention relates generally to the fields of biochemical pharmacology and drug discovery. More specifically, the present invention relates to the novel mRNA capping enzymes Pgt1 and Pgt1 from Plasmodium falciparum, the agent of malaria, and methods of screening for antimalarial and antiprotozoal compounds that inhibit mRNA cap formation.
2. Description of the Related Art
Malaria extracts a prodigious toll each year in human morbidity (400 million new cases) and mortality (1 million deaths). The malaria parasite is transmitted when humans are bitten by the Anopheles mosquito. Of the four species of Plasmodium parasites that cause human malariaxe2x80x94Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium falciparumxe2x80x94it is P. falciparum that is principally responsible for fulminant disease and death. Malaria treatment and prevention strategies have been steadily undermined by the spreading resistance of the Plasmodium pathogen to erstwhile effective drugs and of the mosquito vector to insecticides [1]. Thus, there is an acute need for new malaria therapies.
It is anticipated that the Plasmodium falciparum genome project [2] will uncover novel targets for therapy and immunization. The most promising drug targets will be those gene products or metabolic pathways that are essential for all stages of the parasite life cycle, but either absent or fundamentally different in the human host and the arthropod vector. Such targets can be identified either by whole-genome comparisons or by directed analyses of specific cellular transactions. In those instances where Plasmodium differs from metazoans, comparisons to other unicellular organisms may provide insights into eukaryotic phylogeny.
Processing of eukaryotic mRNA in vivo is coordinated temporally and physically with transcription. The earliest event is the modification of the 5xe2x80x2 terminus of the nascent transcript to form the cap structure m7GpppN. The cap is formed by three enzymatic reactions: (i) the 5xe2x80x2 triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA 5xe2x80x2 triphosphatase; (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase; and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase [3].
RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus that abrogate catalytic activity are lethal in vivo. Genetic and biochemical experiments highlight roles for the cap in protecting mRNA from untimely degradation by cellular 5xe2x80x2 exonucleases and in recruiting the mRNA to the ribosome during translation initiation.
The physical and functional organizations of the capping apparatus differ in significant respects in metazoans, fungi, and viruses. Mammals and other metazoa encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide and a separate methyltransferase polypeptide. Fungi encode a three-component system consisting of separate triphosphatase, guanylyltransferase, and methyltransferase gene products. Viral capping systems are quite variable in their organization; poxviruses encode a single polypeptide containing all three active sites, whereas phycodnaviruses encode a yeast-like capping apparatus in which the triphosphatase and guanylyltransferase enzymes are encoded separately [4].
The guanylyltransferase and methyltransferase components of the capping apparatus are mechanistically conserved between metazoans and budding yeast. In contrast, the structures and catalytic mechanisms of the mammalian and fungal RNA triphosphatases are completely different [5]. The triphosphatase components of many viral mRNA capping enzymes are mechanistically and structurally related to the fungal RNA triphosphatases, and not to the host cell triphosphatase [4, 6, 7]. Thus, cap formation and cap-forming enzymes, especially RNA triphosphatase, are promising targets for antifungal and antiviral drug discovery.
A plausible strategy for antimalarial drug discovery is to identify compounds that block Plasmodium-encoded capping activities without affecting the capping enzymes of the human host or the mosquito vector. For this approach to be feasible, the capping enzymes of the malaria parasite must be identified.
Little is known about the organization of the mRNA capping apparatus in the many other branches of the eukaryotic phylogenetic tree. RNA guanylyltransferase has been studied in the kinetoplastids Trypanosoma and Crithidia [8] but the triphosphatase and methyltransferase components have not been identified.
RNA Guanylyltransferasexe2x80x94Transfer of GMP from GTP to the 5xe2x80x2 diphosphate terminus of RNA occurs in a two-step reaction involving a covalent enzyme-GMP intermediate [3]. Both steps require a divalent cation cofactor.
(i) E+pppG⇄E-pG+PPi
(ii) E-pG+ppRNA⇄GpppRNA+E
The GMP is covalently linked to the enzyme through a phosphoamide (Pxe2x80x94N) bond to the epsilon-amino group of a lysine residue within a conserved KxDG element (motif I) found in all known cellular and DNA virus-encoded capping enzymes (FIG. 1). Five other sequence motifs (III, IIIa, IV, V, and VI) are conserved in the same order and with similar spacing in the capping enzymes from fungi, metazoans, DNA viruses, and trypanosomes (FIG. 1) [3].
H{dot over (a)}kansson et al. [9] have determined the crystal structure of the Chlorella virus guanylyltransferase in the GTP-bound state and with GMP bound covalently. The protein consist of a larger N-terminal domain (domain 1, containing motifs I, III, IIIa, and IV) and a smaller C-terminal domain (domain 2, containing motif VI) with a deep cleft between them. Motif V bridges the two domains. Motifs I, III, IIIa, IV, and V form the nucleotide binding pocket. The crystal structure reveals a large conformational change in the GTP-bound enzyme, from an xe2x80x9copenxe2x80x9d to a xe2x80x9cclosedxe2x80x9d state, that brings motif VI into contact with the beta and gamma phosphates of GTP and reorients the phosphates for in-line attack by the motif I lysine.
Identification of essential amino acids has been accomplished by site-directed mutagenesis of Cegt1, the RNA guanylyltransferase of Saccharomyces cerevisiae. The guanylyltransferase activity of Cegt1p is essential for cell viability. Hence, mutational effects on Ceg1 function in vivo can be evaluated by simple exchange of mutant CEG1 alleles for the wild type gene. The effects of alanine substitutions for individual amino acids in motifs I, III, IIIa, IV, V, and VI have been examined. Sixteen residues were defined as essential (denoted by dots in FIG. 1) and structure-activity relationships at these positions were subsequently determined by conservative replacements [10]. Many of the essential Cegt1 side chains correspond to moieties which, in the Chlorella virus capping enzyme crystal structure, make direct contact with GTP as denoted by the arrowheads in FIG. 1.
RNA Triphosphatasexe2x80x94There are at least two mechanistically and structurally distinct classes of RNA 5xe2x80x2 triphosphatases: (i) the divalent cation-dependent RNA triphosphatase/NTPase family (exemplified by Saccharomyces cerevisiae Cet1 and Cth1, Candida albicans CaCet1, Schizosaccharomyces pombe Pct1, Chlorella virus Rtp1, baculovirus LEF-4, and vaccinia virus D1), which require three conserved collinear motifs (A, B, and C) for activity [4,6,7,11-14], and (ii) the divalent cation-independent RNA triphosphatases, e.g., the metazoan cellular mRNA capping enzymes, the baculovirus phosphatase BVP, and the human enzyme PIR1, which require a HCxxxxxR(S/T) phosphate-binding motif [15-17].
Metazoan capping enzymes consist of an N-terminal RNA triphosphatase domain and a C-terminal guanylyltransferase domain. In the 497-amino acid mouse enzyme Mce1, the two catalytic domains are autonomous and nonoverlapping [15]. The metazoan RNA triphosphatases belong to a superfamily of cysteine phosphatases that includes protein tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases. The metazoan RNA triphosphatases contain a HCxxxxxR(S/T) signature motif (referred to as the P loop) that defines the cysteine phosphatase superfamily (FIG. 2). Metazoan RNA triphosphatases catalyze the cleavage of the xcex3 phosphate of 5xe2x80x2 triphosphate RNA via a two-step pathway. First, a cysteine thiolate nucleophile of the enzyme (the conserved cysteine of the P loop) attacks the xcex3 phosphorus to form a covalent protein-cysteinyl-S-phosphate intermediate [16] and release the diphosphate-terminated product. Then the covalent intermediate is hydrolyzed to liberate inorganic phosphate. The metazoan RNA triphosphatases do not require a metal cofactor.
Saccharomyces cerevisiae Cet1 exemplifies the class of divalent cation-dependent RNA triphosphatase enzymes, which includes the RNA triphosphatase encoded by the pathogenic fungus Candida albicans, the fission yeast Schizosaccharomyces pombe, and the RNA triphosphatase components of the capping systems of poxviruses, baculoviruses, and Chlorella virus PBCV-1. This triphosphatase family is defined by three conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids that are essential for Cet1 catalytic activity [6,12] (FIG. 3).
Purified recombinant Cet1 catalyzes the magnesium-dependent hydrolysis of the xcex3 phosphate of triphosphate-terminated RNA to form a 5xe2x80x2 diphosphate end. Cet1 also displays a robust ATPase activity in the presence of manganese or cobalt, but magnesium, calcium, copper, and zinc are not effective cofactors for ATP hydrolysis [6]. Cet1 displays broad specificity in converting rNTPs and dNTPs to their respective diphosphates. The manganese- and cobalt-dependent NTPase activity of Cet1 resembles the manganese- or cobalt-dependent NTPase activities of the of the other members of this family, including baculovirus LEF-4, C. albicans CaCet1, S. cerevisiae Cth1, S. pombe Pct1, and Chlorella virus Rtp1 [4,11-14].
Crystal Structure of Fungal RNA Triphosphatasexe2x80x94The biologically active triphosphatase derivative Cet1(241-539) was crystallized and its structure determined at 2.05 xc3x85 resolution [5]. Consistent with solution studies, Cet1 crystallized as a dimer. The striking feature of the tertiary structure is the formation of a topologically closed tunnel composed of 8 antiparallel xcex2 strands. The active site resides within this hydrophilic xe2x80x9ctriphosphate tunnelxe2x80x9d. The xcex2 strands that comprise the walls of the tunnel are displayed over the Cet1 protein sequence in FIG. 3. The interior of the tunnel contained a single sulfate ion coordinated by two arginine and two lysine side chains. Insofar as sulfate is a structural analog of phosphate, it is likely that the side chain interactions of the sulfate reflect contacts made by the enzyme with the xcex3 phosphate of the triphosphate-terminated RNA and nucleoside triphosphate substrates.
The proteins most closely related to Cet1 at the primary structure level are CaCet1, Pct1, and Cth1. CaCet1 is the RNA triphosphatase component of the capping apparatus of Candida albicans. Pct1 is the RNA triphosphatase component of the capping apparatus of Schizosaccharomyces pombe [14]. Cth1 is a nonessential S. cerevisiae protein with divalent cation-dependent RNA triphosphatase/NTPase activity that may participate in an RNA transaction unrelated to capping [12]. The amino acid sequences of Cet1, CaCet1, Pct1, and Cth1 are aligned in FIG. 3. The residues conserved in all four fungal enzymes are localized predominantly in the interior of the tunnel.
Cet1 triphosphatase activity is strictly dependent on a divalent cation cofactor. The hydrolysis of 5xe2x80x2 triphosphate RNA termini is optimal in the presence of magnesium, whereas NTP hydrolysis specifically requires manganese or cobalt. The location of a metal-binding site on the enzyme was determined by X-ray diffraction of Cet1(241-539) crystals that had been soaked in manganese chloride [5]. Manganese is coordinated with octahedral geometry to the sulfate inside the tunnel, to the side chain carboxylates of three glutamates, and to two waters. The three glutamates that comprise the metal-binding site of fungal RNA triphosphatase are located in motifs A and C, which define the metal-dependent RNA triphosphatase family. Substitution of any one of the three glutamates by alanine or glutamine inactivates Cet1. The motif A and C glutamates are also essential for the activities of vaccinia virus RNA triphosphatase, baculovirus RNA triphosphatase, C. albicans CaCet1, S. pombe Pct1, and S. cerevisiae Cth1. Thus, it is likely that motifs A and C comprise the metal binding site in all members of this enzyme family.
The structure of Cet1(241-539) with bound sulfate and manganese is construed to reflect that of the product complex of enzyme with the hydrolyzed xcex3 phosphate [5]. The structure suggests a catalytic mechanism whereby acidic side chains located on the floor of the tunnel coordinate an essential divalent cation that in turn coordinates the xcex3 phosphate. The metal ion would activate the xcex3 phosphorus for direct attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the xcex2 phosphate leaving group. Interactions between the sulfate and basic side chains located on the walls of the tunnel would contribute to the coordination of the 5xe2x80x2 phosphates in the ground state and the stabilization of the negative charge on the xcex3 phosphate developed in the transition state. A key mechanistic distinction between the fungal-type RNA triphosphatases and the metazoan-type RNA triphosphatases is that the fungal-type enzymes do not form a covalent phosphoenzyme intermediate.
The prior art is deficient in the lack of methods that teach a person having ordinary skill in this art how to screen for a compound that inhibits cap formation by the enzymes of unicellular eukaryotic parasites such as Plasmodia. The prior art is also deficient in an identification and characterization of the enzymes comprising the mRNA capping apparatus of Plasmodia. In particular, the RNA triphosphatase component of the mRNA capping apparatus has not been identified and characterized in any unicellular eukaryotic parasite. The biochemical properties of an RNA triphosphatase from a unicellular eukaryotic parasite are not known. Hence, a mechanistic and structural comparison between the RNA triphosphatase of the parasite and the RNA triphosphatase of the metazoan host organism, which could underscore the potential of RNA triphosphatase as a therapeutic target for parasitic infections, is not possible. The present invention fulfills this longstanding need in the art.
The present invention facilitates the discovery of drugs that target an essential aspect of gene expressionxe2x80x94the formation of the mRNA 5xe2x80x2 cap m7GpppNxe2x80x94in unicellular eukaryotic parasites.
The invention discloses the amino acid sequences of the Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase, which catalyze the first and second steps of mRNA cap formation, respectively. The invention also provides for expression vectors and recombinant Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase.
The invention further encompasses in vitro screening methods to identify candidate inhibitors of the catalytic activity of RNA guanylyltransferase or the RNA 5xe2x80x2 triphosphatase of unicellular eukaryotic parasites. These methods are simple, quantitative, and adaptable to colorimetric, spectrophotometric, or fluorescence detection assays that are suited to high-throughput screening for inhibitors of the RNA triphosphatase of Plasmodia and other unicellular eukaryotic parasites.